M5076 Tumor Research Articles

Abstract 92: Deploying spatial transcriptomics to inform intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples

Abstract Uveal Melanoma (UM) is a rare tumor characterized by mutually-exclusive activating mutations in GNAQ or GNA11, followed by a mutation event in BAP1, SF3B1 or EIF1AX. Notably, a large subset of patients present with a unique chromosome 3 copy-loss event, which is highly associated with metastatic progression in late-stage disease. To date, the biology underlying the causes and consequences of this unique copy-loss event (Monosomy 3 or M3), and its role in promoting metastases remains largely unexplored. This can be attributed to both the lack of preclinical models and in-depth characterization of paired primary and metastatic tumors in patients. To address this, we derived a preclinical isogenic model of chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Isogenic Disomy (D3) and Monosomy (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired, in vivo, primary and metastatic settings. This modeling has enabled us to employ spatial transcriptomics in a PDX setting to characterize gene expression signatures across unique D3 and M3 UM clones. Investigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage). We have also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, where we are able to overcome the lack of same-species microenvironment controls. Our methodology allows for deep characterization of sub-clonal heterogeneity in both D3 and M3 settings and informs on the unique biology underlying invasiveness and outgrowth of M3 tumors. Finally, we complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors. More broadly, comparing these D3 vs. M3 signatures provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival. Citation Format: Sanjana Srinivasan, Johnathon Rose, Joseph R. Daniele, Michael Peoples, Meng He, Rosalba Minelli, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Khalida Wani, Luigi Perelli, Dmitriy Loza, Ningping Feng, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, P. Andrew Futreal, Scott E. Woodman, Alexander J. Lazar, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Deploying spatial transcriptomics to inform intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 92.

A Therapeutic Vaccine in Combination with Cyclic GMP-AMP Cures More Differentiated Melanomas in Mice.

We have identified a combinational immunotherapy termed TheraVac vaccine (TheraVac) that can cure multiple large established mouse tumors, but it failed to cure melanoma in mice. TheraVac consists of an immunostimulating arm containing an agonist (HMGN1 [N1]) for TLR4 and an agonist (R848) for TLR7/8 that synergize to activate tumor-infiltrating dendritic cells (DCs) and promote Th1 immune responses. The second arm uses an immune checkpoint blockade, anti-PDL-1, to diminish tumor-associated immunosuppression. In this study, we investigated supplementation of TheraVac by a stimulator of IFN genes (STING) agonist, cyclic GMP-AMP (cGAMP), because together they synergize in activating DCs and produced more immunostimulating IL-12p70 and TNF-α cytokines. The synergistic activation and maturation of DCs is dependent on the activation of tank binding kinase-1 (TBK1). Treatment of three different melanin-producing mouse melanomas (B16F1, M3, and M4) with intratumoral delivery of cGAMP and TheraVac eradicated 60-80% of these melanomas. Immunoprofiling of M3 tumor treated with TheraVac plus cGAMP showed an increase in CD8+ CTLs and macrophages in the tumor. There was also a marked increase of CD4, CD8 effector and memory T cells and generation of functional tumor-specific CTLs in tumor-draining lymph nodes. The resultant tumor-free mice were selectively resistant to subsequent challenge with the same tumors, indicating long-term tumor-specific protective immunity. Overall, our findings have important implications for clinical trials with a combination of these immunotherapeutics to cure melanin-producing human melanomas, without the need for exogenous tumor Ags and no clear toxic effects in mice.

68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice.

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvβ3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvβ3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.

Therapeutic Vaccine (TheraVac) in combination with cGAMP cures more differentiated melanomas in mice

Abstract We have identified a combination of immunotherapeutics, termed TheraVac, capable of curing multiple large established mouse tumors, but it failed to cure melanoma in mice. TheraVac consists of an immunostimulating arm containing an agonist (HMGN1) for TLR4 and an agonist (R848) for TLR7/8 that synergize to activate tumor-infiltrating dendritic cells (DCs) and upregulate the polarization of Th1 immune responses. The second arm uses an immune checkpoint blockade inhibitor, αPDL-1, to diminish tumor associated immunosuppression. Here, we investigated supplementation of TheraVac by a STING agonist, cGAMP, because they synergize in activating DC’s and produced more immunostimulating IL-12p70 and TNFα cytokines. We therefore treated five different tumor types including melanin producing B16F1, M3, and M4 mouse melanomas that are homologous to human melanomas with intratumoral delivery of cGAMP and TheraVac. This eradicated 80% of the B16F1, 80% of the M3 and 60% of the M4 tumors, but still failed to cure the non-melanin producing undifferentiated M1 and M2 tumors. Biopsies of M3 tumor treated with TheraVac plus cGAMP showed a significant increase of CD8+ CTL’s infiltrating into the tumor. Importantly, there was a marked increase of both CD4 and CD8 memory T cells and generation of functional tumor specific CTLs in tumor-draining lymph nodes. The resultant tumor-free mice were selectively resistant to subsequent challenge with the same tumors, indicating long-term tumor specific protective immunity. Overall, our findings have important implications for clinical trials with a combination of immunotherapeutics to cure melanin producing human melanomas, without the need for exogenous tumor antigens and no obvious toxic effects in mice.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

We have developed a fully synthetic and multifunctional antibody‐recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre‐immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo‐RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αvβ3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.

New Folate-Grafted Chitosan Derivative To Improve Delivery of Paclitaxel-Loaded Solid Lipid Nanoparticles for Lung Tumor Therapy by Inhalation.

Inhaled chemotherapy for the treatment of lung tumors requires that drug delivery systems improve selectivity for cancer cells and tumor penetration and allow sufficient lung residence. To this end, we developed solid lipid nanoparticles (SLN) with modified surface properties. We successfully synthesized a new folate-grafted copolymer of polyethylene glycol (PEG) and chitosan, F-PEG-HTCC, with a PEG-graft ratio of 7% and a molecular weight range of 211-250 kDa. F-PEG-HTCC-coated, paclitaxel-loaded SLN were prepared with an encapsulation efficiency, mean diameter, and zeta potential of about 100%, 250 nm, and +32 mV, respectively. The coated SLN entered folate receptor (FR)-expressing HeLa and M109-HiFR cells in vitro and M109 tumors in vivo after pulmonary delivery. The coated SLN significantly decreased the in vitro half-maximum inhibitory concentrations of paclitaxel in M109-HiFR cells (60 vs 340 nM, respectively). We demonstrated that FR was involved in these improvements, especially in M109-HiFR cells. After pulmonary delivery in vivo, the coated SLN had a favorable pharmacokinetic profile, with pulmonary exposure to paclitaxel prolonged to up to 6 h and limited systemic distribution. Our preclinical findings therefore demonstrated the positive impact of the coated SLN on the delivery of paclitaxel by inhalation.

Overcoming resistance to anti-PD immunotherapy in a syngeneic mouse lung cancer model using locoregional virotherapy

ABSTRACTAnti-PD-1 and anti-PD-L1 immunotherapy has provided a new therapeutic opportunity for treatment of advanced-stage non-small cell lung cancer (NSCLC). However, overall objective response rates are approximately 15%–25% in all NSCLC patients who receive anti-PD therapy. Therefore, strategies to overcome primary resistance to anti-PD immunotherapy are urgently needed. We hypothesized that the barrier to the success of anti-PD therapy in most NSCLC patients can be overcome by stimulating the lymphocyte infiltration at cancer sites through locoregional virotherapy. To this end, in this study, we determined combination effects of anti-PD immunotherapy and oncolytic adenoviral vector-mediated tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) gene therapy (Ad/E1-TRAIL) or adenoviral-mediated TP53 (Ad/CMV-TP53) gene therapy in syngeneic mice bearing subcutaneous tumors derived from M109 lung cancer cells. Both anti–PD-1 and anti–PD-L1 antibodies failed to elicit obvious therapeutic effects in the M109 tumors. Intratumoral administration of Ad/E1-TRAIL or Ad/CMV-TP53 alone suppressed tumor growth in animals preexposed to an adenovector and bearing subcutaneous tumors derived from M109 cells. However, combining either anti–PD-1 or anti–PD-L1 antibody with these two adenoviral vectors elicited the strongest anticancer activity in mice with existing immunity to adenoviral vectors. Dramatically enhanced intratumoral immune response was detected in this group of combination therapy based on infiltrations of CD4+ and CD8+ lymphocytes and macrophages in tumors. Our results demonstrate that resistance to anti–PD-1 immunotherapy in syngeneic mouse lung cancer can be overcome by locoregional virotherapy.

Targeting tumors with cyclic RGD-conjugated lipid nanoparticles loaded with an IR780 NIR dye: In vitro and in vivo evaluation

Like several 50nm-large nanocarriers, lipid nanoparticles (LNPs) can passively accumulate in tumors through the Enhanced Permeability and Retention (EPR) effect. In this study, we developed PEGylated LNPs loaded with IR780 iodide as a contrast agent for NIR fluorescence imaging and modified them with cyclic RGD peptides in order to target integrin avβ3. We demonstrate a specific targeting of the receptor with cRGD-LNPs but not with cRAD-LNP and standard LNP using HEK293(β3), HEK293(β3)-αvRFP, DU145 and PC3 cell lines. We also demonstrate that cRGD-LNPs bind to αvβ3, interfere with cell adhesion to vitronectin and co-internalize with αvβ3 within one hour. We then investigated their biodistribution and tumor targeting in mice bearing DU145 or M21 tumors. We observed no significant differences between cRGD-LNP and the non-targeted ones regarding their biodistribution and accumulation/retention in tumors. This suggested that despite an efficient formulation of the cRGD-LNPs, the cRGD-mediated targeting was not increasing the total amount of LNP that can already accumulate passively in the subcutaneous tumors via the EPR effect.

Tissue distribution and tumor uptake of folate receptor–targeted epothilone folate conjugate, BMS-753493, in CD2F1 mice after systemic administration

To assess targeting of an epothilone folate conjugate (BMS-753493) to the folate receptor (FR)-overexpressed tumor in mice bearing both FR+ and FR– tumors, a series of experiments were conducted by quantitative whole-body autoradiography (QWBA) and LC–MS/MS following i.v. administration of BMS-753493 or its active moiety, BMS-748285 in mice bearing FR+ (98M109) and FR– (M109) tumors. QWBA showed [3H]BMS-753493–derived radioactivity was extensively distributed to various tissues. The FR over-expressing 98M109 tumors showed consistently higher level of radioactivity than FR-negative tumors (i.e., M109 tumors) up to 48h post dose of [3H]BMS-753493, despite the magnitude of difference between the tumors is relatively small (generally 3~5-fold). The radioactivity level in 98M109 tumors was 2~12-fold of normal tissues except intestine/content at 48h post dose. No selective radioactivity uptake into 98M109 tumors over M109 or normal tissues was observed after i.v. administration of the active epothilone, [3H]BMS-748285. LC–MS/MS measurements demonstrated that the concentrations of BMS-748285, presumably from hydrolysis of the folate conjugate, in 98M109 tumors were greater than those in M109 tumors after i.v. administration of BMS-753493 (2–3-fold) whereas no differential uptake in the tumors following BMS-748285 administration. Those data were consistent with radioactivity determinations. Those results demonstrated that the folate conjugation in BMS-753493 enabled moderately preferential distribution of the active epothilone to FR over-expressing 98M109 tumors, thereby supporting targeted delivery of cytotoxics through the folate receptor.

Variation of Specific Activities of 68Ga-Aquibeprin and 68Ga-Avebetrin Enables Selective PET Imaging of Different Expression Levels of Integrins α5β1 and αvβ3.

Fully automated 68Ga labeling of 0.3 nmol of aquibeprin or avebetrin was done using buffered eluate fractions (600-800 MBq, pH 2) of an SnO2-based generator, affording the radiopharmaceuticals with specific activities greater than 1,000 MBq/nmol. Lower values ranging from 150 to 0.4 MBq/nmol were adjusted by addition of inactive compound (∼0.15-50 nmol) to the injected activity (∼20 MBq for PET, 5-7 MBq for biodistribution). For in vivo experiments, 6- to 12-wk-old female severe combined immunodeficiency mice bearing M21 xenografts (human melanoma, expressing both integrins α5β1 and αvβ3) were used. The expression density of integrin β3 was determined by immunohistochemistry on paraffin slices. For mass doses (specific activities) of less than 20 pmol (>1,000 MBq/nmol) and 1 nmol (20 MBq/nmol) per mouse, respectively, uptake of 68Ga-aquibeprin and 68Ga-avebetrin in M21 tumors dropped from 5.3 and 3.5 to 3.0 and 2.4 percentage injected dose per gram (%ID/g), respectively. When less than 20 pmol was applied, high uptake of 68Ga-aquibeprin in the eyes (4.5 %ID/g) or 68Ga-avebetrin in adrenals (25.9 %ID/g), respectively, were found, which was reduced by 90% and 65% (0.44 and 6.2 %ID/g, respectively), for doses of 1 nmol. The highest tumor-to-tissue ratios were observed both in ex vivo biodistribution and PET for comparably large doses, for example, 6 nmol (0.65 mg/kg) 68Ga-aquibeprin per mouse (3.5 MBq/nmol). Presumably because of their high affinities, 68Ga-aquibeprin and 68Ga-avebetrin allow for selective addressing of target sites with different integrin expression levels by virtue of adjusting specific activity, which can be exploited for visualization of low-level target expression or optimization of tumor-to-background contrast.

Complementary, Selective PET Imaging of Integrin Subtypes α5β1 and αvβ3 Using 68Ga-Aquibeprin and 68Ga-Avebetrin.

Despite in vivo mapping of integrin αvβ3 expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α5β1 appears to be a promising target, for which purpose we designed the PET radiopharmaceutical (68)Ga-aquibeprin. (68)Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α5β1 integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated (68)Ga labeling. Integrin α5β1 and αvβ3 affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α5 and β3 subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. (68)Ga-aquibeprin shows high selectivity for integrin α5β1 (50% inhibition concentration [IC50] = 0.088 nM) over αvβ3 (IC50 = 620 nM) and a pronounced hydrophilicity (log D = -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the β3 integrin subunit but also an intense expression of the α5 integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. (68)Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for (68)Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer (68)Ga-avebetrin, which shows an inverse selectivity for integrin αvβ3 (IC50 = 0.22 nM) over α5β1 (IC50 = 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (∼5 mg/kg) of the respective other compound. (68)Ga-aquibeprin and (68)Ga-avebetrin are recommendable for complementary mapping of integrins α5β1 and αvβ3 by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.

Chemotherapy in vivo against M109 murine lung carcinoma with cytochalasin B by localized, systemic, and liposomal administration.

Cytochalasin B is a potentially novel microfilament-directed chemotherapeutic agent that prevents actin polymerization, thereby inhibiting cytokinesis. Although cytochalasin B has been extensively studied in vitro, only limited data are available to assess its in vivo potential. Cytochalasin B was administered to Balb/c mice challenged i.d. with M109 murine lung carcinoma to determine whether the agent could affect an established i.d. tumor when the compound is administered s.c. in the region of the i.d. tumor, but not in direct contact with it. Cytochalasin B was also administered either i.p. or s.c. at a distant site or i.v. to determine whether it could affect the long-term development of an established i.d. tumor. Cytochalasin B was then liposome encapsulated to determine whether the maximum tolerated dose (MTD) of the compound could be increased, while reducing immunosuppression that we have previously characterized. Liposomal cytochalasin B was also administered to mice challenged i.d. with M109 lung carcinoma to assess its chemotherapeutic efficacy. The results can be summarized as follows: 1) cytochalasin B substantially delayed the growth of i.d. M109 tumor nodules, inhibited metastatic progression in surrounding tissues, and produced long-term cures in treated mice; 2) liposomal cytochalasin B increased the i.p. MTD by more than 3-fold, produced a different distribution in tissue concentrations, and displayed antitumor effects against M109 lung carcinoma similar to non-encapsulated cytochalasin B. These data show that cytochalasin B exploits unique chemotherapeutic mechanisms and is an effective antineoplastic agent in vivo in pre-clinical models, either in bolus form or after liposome encapsulation.

Benefits of NOPO As Chelator in Gallium-68 Peptides, Exemplified by Preclinical Characterization of68Ga-NOPO–c(RGDfK)

The αvβ3-integrin addressing cyclic pentapeptide cyclo(RGDfK) was conjugated to NOPO, 1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid], a bifunctional chelator with exceptional gallium-68 labeling properties. NOPO-c(RGDfK) and its Ga(III) and Cu(II) complexes showed high affinity to αvβ3 integrin (IC50 = 0.94 ± 0.06, 1.02 ± 0.09, and 0.51 ± 0.06 nM, respectively). (68)Ga labeling of NOPO-c(RGDfK) in an automated GMP-compliant procedure was performed with near-quantitative radiochemical yield, using precursor amounts as low as 0.5 nmol (approximately 0.6 μg). (68)Ga-NOPO-c(RGDfK) was obtained with high purity (>99% by radio-HPLC/TLC) and, optionally, could be produced with specific activities up to 6 TBq/μmol. M21/M21L (human melanoma with high/low αvβ3 integrin expression) xenografted athymic CD-1 nude mice were used for biodistribution, in vivo stability studies, and PET imaging. (68)Ga-NOPO-c(RGDfK) showed rapid and specific uptake in M21 tumor xenografts (2.02 ± 0.34% ID/g at 60 min p.i.) and was found stable in vivo. Its high hydrophilicity is reflected by an octanol-water distribution coefficient (log D = -4.6) which is more than 1 order of magnitude lower compared to respective NOTA or DOTA analogues. As expected, (68)Ga-NOPO-c(RGDfK) thus showed fast renal clearance from nontargeted tissues. We conclude that NOPO might generally prove a useful means to improve renal clearance of corresponding radiopharmaceuticals by increasing the polarity of its bioconjugates. Favorable labeling properties render NOPO conjugates highly recommendable for reliable routine production of (68)Ga-radiopharmaceuticals in a clinical setting.

Imaging of firefly luciferase activity under antiangiogenic therapy in a heat shock protein 70-transfected melanoma tumor model.

We investigated the effect of targeted gene therapy on heat shock protein 70 (Hsp70) expression in a melanoma tumor model (M21). M21 cells transfected with a plasmid containing the firefly luciferase reporter gene (ffluc), whose expression is driven by the hsp70 (hspa1b) or the cytomegalovirus (CMV) promoter, were grown to a size of 600 mm3. Five animals in each group were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein [RGD-NP/RAF(-)] complex. Bioluminescence imaging (BLI) (IVIS, Xenogen, Alameda, CA) was performed at set time intervals. Western blot analysis of the HSP70 protein was simultaneously performed. The size of the treated M21 tumors was nearly constant (637.8 ± 33.4 mm3 vs 674.8 ± 34.4 mm3). BLI showed that if transcription was controlled by the CMV promoter, firefly luciferase activity decreased to 51.1% ± 8.3%. When transcription was controlled by the hsp70 promoter, the highest firefly luciferase activity (4.4 ± 0.3-fold) was observed after 24 hours. In accordance with BLI, Western blot analysis showed an increase in the level of HSP70, with the maximum detection 24 hours after the injection of the RGD-NP/RAF(-) complex. Targeted antiangiogenic therapy can induce luciferase activity where transcription is controlled by an hsp70 promoter and HSP70 protein in melanoma tumors.

64Cu-p-NH2-Bn-DOTA-hu14.18K322A, a PET Radiotracer Targeting Neuroblastoma and Melanoma

The hu14.18K322A variant of the GD2-targeting antibody hu14.18 has been shown to elicit a level of antibody-dependent cell-mediated cytotoxicity toward human neuroblastoma cells similar to that of the parent antibody. However, hu14.18K322A exhibited a decreased complement activation and associated pain, the dose-limiting toxicity in neuroblastoma immunotherapy. PET with a radiolabeled analog of the same antibody used in treatment will provide insight into the ability of hu14.18K322A to reach its target, as well as nontarget uptake that may cause side effects. Such antibody radiotracers might also provide a method for measuring GD2 expression in tumors, thus enabling the prediction of response to anti-GD2 therapy for individual patients. The conjugation of hu14.18K322A with p-NH(2)-Bn-DOTA was accomplished using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with subsequent (64)Cu radiolabeling at 37°C for 30 min. Immunoreactivity of the conjugate was assessed by a dose-escalation blocking experiment measuring binding to purified GD2 versus GD1b as a negative control. Cell uptake and biodistribution studies in M21 (GD2-positive) and PC-3 (GD2-negative) tumor models were performed, as was small-animal PET/CT of M21 and PC-3 tumor-bearing mice. The labeling of (64)Cu-p-NH(2)-Bn-DOTA-hu14.18K322A was achieved at more than 95% radiochemical purity and a specific activity of 127-370 MBq/mg (3.4-10 mCi/mg) after chromatographic purification. Preliminary in vitro data demonstrated a greater than 6-fold selectivity of binding to GD2 versus GD1b and dose-dependent inhibition of binding by unmodified hu14.8K322A. In vivo data, including small-animal PET/CT, showed significant GD2-positive tumor-targeting ability, with a persistent 2-fold-higher uptake of radiotracer than in GD2-negative tumors. (64)Cu-p-NH(2)-Bn-DOTA-hu14.18K322A represents a novel PET radiotracer to facilitate clinical investigations of anti-GD2 immunotherapies and to complement other imaging modalities in the staging and treatment of neuroblastoma.

Be spoilt for choice with radiolabelled RGD peptides: Preclinical evaluation of 68Ga-TRAP(RGD)3

Gallium-68 is rapidly gaining importance, as this generator-produced PET isotope is available independent of on-site cyclotrons, enabling radiopharmaceutical production with comparably simple techniques at low cost. The recently introduced TRAP chelator combines the advantage of straightforward design of multimeric ⁶⁸Ga-radiopharmaceuticals with very fast and efficient ⁶⁸Ga-labeling. We synthesized a series of five cyclo(RGDfK) peptide trimers and determined their α(v)β₃ integrin affinities in competition assays on α(v)β₃-expressing M21 human melanoma cells against ¹²⁵I-echistatin. The compound with highest IC₅₀, Ga-TRAP(RGD)₃, showed more than 7-fold higher affinity compared to the monomers F-Galacto-RGD and Ga-NODAGA-c(RGDyK). TRAP(RGD)₃ was radiolabeled with ⁶⁸Ga in a fully automated GMP compliant manner. CD-1 athymic nude mice bearing M21/M21L human melanoma xenografts were used for biodistribution studies, blockade experiments, metabolite studies and PET imaging. ⁶⁸Ga-TRAP(RGD)₃ exhibited high M21 tumor uptake (6.08±0.63% ID/g, 60 min p.i.), was found to be fully stable in vivo, and showed a fast renal clearance. Blockade studies showed that uptake in the tumor, as well as in all other tissues, is highly integrin specific. A comparison of biodistribution and PET data of ⁶⁸Ga-TRAP(RGD)₃ with those of ⁶⁸Ga-NODAGA-c(RGDyK) and ¹⁸F-Galacto-RGD showed that the higher affinity of the trimer effects a larger dynamic response of tracer uptake to integrin expression, i.e., enhanced integrin-specific uptake in all tissues. We conclude that ⁶⁸Ga-TRAP(RGD)₃ could allow for imaging of low-level integrin expression in tissues which are not visible with the two competitors. Overall, the study constitutes proof of concept for the favourable in vivo properties of TRAP-based ⁶⁸Ga radiopharmaceuticals.

In vivo monitoring of antiangiogenic therapy by magnetic resonance and bioluminescence imaging in an M21 tumor model through activation of an hsp70 promoter-luciferase reporter construct

We have investigated the effect of targeted gene therapy on the melanoma cell line M21, using a combination of bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). M21 cells transfected with a plasmid containing either an hsp70 (Hspa1b) or a CMV promoter fragment, along with the luciferase reporter gene, were grown to a tumor size of 900 mm(3) . Five mice in each group were intravenously treated every 72 h with a complex consisting of a nanoparticle, an Arg-Gly-Asp-peptide, and a dominant negative mutant protein kinase inhibitor gene. BLI and MRI were performed at specific time intervals. The MRI scan protocol included T(1) -weighted-spin-echo ± contrast medium, T(2) -weighted-fast-spin-echo, dynamic contrast-enhanced MRI (DCE-MRI), and diffusion-weighted-stimulated-echo-acquisition-mode-sequence. The T(2) times were obtained using a 1.5 T GE MRI scanner. The size of the treated M21 tumors remained almost constant during the treatment phase (837.8 ± 133.4 vs 914.8 ± 134.4 mm(3) ). BLI showed that, if transcription was controlled by the CMV promoter, the luciferase activity decreased to 51.1 ± 8.3%. After transcription was controlled by the hsp70 promoter, the highest luciferase activity (4.4 ± 0.3 fold) was seen after 24 h. The signal-to-noise ratio (SNR; T(2) -weighted images) of the tumors was 36.7 ± 0.6 and subsequently dropped to 31.2 ± 4.4 (p=0.004). DCE-MRI showed a reduction of the slope and the Ak(ep) of 67.8% ± 4.3 and 64.8% ± 3.3%, respectively, compared with the baseline. The SNR value (T(1) -weighted images) of the tumors was 42.3 ± 1.9 immediately following contrast medium application and subsequently dropped to 28.5 ± 3.0 (p<0.001). In the treatment group, the diffusion coefficient increased significantly under therapy (0.66 ± 0.05 vs the pretreatment value of 0.54 ± 0.009 p<0.01). Thus, we observed that targeted antiangiogenic therapy can induce activation of the hsp70 promoter through a heat shock/luciferase reporter system. Moreover, MRI showed a significant reduction of the contrast medium uptake parameters and an increase in the diffusion coefficient of the tumors.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm(3). Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8 ± 103.4 mm(2) at the beginning versus 704.8 ± 94.4 mm(3) at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9% ± 4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5 ± 0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

Use of in vivo bioluminescence and MRI to determine hyperthermia-induced changes in luciferase activity under the control of an hsp70 promoter

We investigated the in vivo effect of hyperthermia on the expression of heat shock proteins and MRI changes in three tumor cell lines. Three tumor cell lines (SCCVII, NIH3T3, M21) were transfected with a plasmid containing the heat shock protein 70 gene (hsp70) promoter fragment and the luciferase reporter gene, and injected into mice. Tumors of 1100 mm³ in size were exposed to five different temperatures (38, 40, 42, 44 and 46 °C) in a water bath. Bioluminescence and MRI were performed at set time intervals. The MRI scan protocol was as follows: T₁-weighted spin echo ± contrast medium, T₂-weighted fast spin echo, dynamic contrast-enhanced MRI, diffusion-weighted stimulated echo acquisition mode sequence, T₂ time obtained on a 1.5T General Electric MRI scanner. Immunoblotting was also performed. hsp70 transcription was strongly induced at 42 and 44 °C, reaching values as high as 8531.5 ± 432.1-fold above baseline in NIH3T3 tumors. At these temperatures, significant increases in the uptake of contrast medium, slope of initial enhancement, Ak(ep) values and apparent diffusion coefficient (ADC) were observed in the 8-h scan of the NIH3T3 cell line. In SCCVII tumors, ADC increased by about 23% (p = 0.010) in the scans performed at 8, 24, 48 and 96 h. At 46 °C, luciferase activity was reduced significantly in the three cell lines. In all tumor types, a significant increase in ADC was observed, which was highest in SCCVII tumors (33.8%; p < 0.01). In accordance with the bioluminescence results, significant Hsp70 protein production was shown by immunoblot analysis. The best correlation coefficient between luciferase activity and immunoblotting results was found for M21 tumors (r = 0.93, p < 0.0001). Different tissue types display distinct patterns of hsp70 transcription. MRI can be used, in combination with optical imaging, to provide information on hsp70 transcription and protein production. The major finding of the present study was that heat-related biochemical changes in tumor tissue can be determined by MRI.

Anti-tumor and immunomodulatory activity of resveratrol in vitro and its potential for combining with cancer immunotherapy

We evaluated the anti-tumor effect of Resveratrol (RV) on M21 and NXS2 tumor cell lines and its immunosuppressive activity on human and murine immune cells to determine the potential for combining RV and immunotherapy. In vitro, concentrations of RV≥25mcM, inhibited cell proliferation, blocked DNA synthesis and induced G1 phase arrest in tumor and immune cells. RV at 12–50mcM inhibited antibody dependent cell mediated cytotoxicity (ADCC) of tumor cells facilitated by the hu14.18-IL2 immunocytokine (IC). The in vivo anti-tumor and immunomodulating activity of RV given systemically were assessed in mice. Results showed that this RV regimen inhibited the growth of NXS2 tumors in vivo but did not appear to interfere with blood cell count, splenocyte or macrophage function. Thus, RV may be a candidate for combining with immunotherapy.